Mini-Trypsin Digestion

Trippin for Trypsin

It’s #TrypsinTuesday! The purpose of this step is to breakdown, or digest, our proteins into peptides.

After talking with Emma yesterday, we realized the protocol we’ve been following is specific for larvae and not oyster tissue. Because of this, the protein concentrations we obtained yesterday were too dilute. To compensate for this, we’re only using sample with 30 µg for 100 µL total volume. Additionally, we needed to modify the protocol for the amount of enzymes used (Lys-C and Trypsin). We divided the amount in the original protocol (made for 100µg protein for 100 µL sample) by three to account for the change in our methods.

Here are the protein concentrations we calculated in after the BCA assay:

Table 1: Volume of sample and 50mM in 6M Urea needed. Sample volume for 30 µg of protein were rounded up to the nearest whole number.

Sample

µL of Sample Required For 30µg Protein

µL of 50mM NH4HCO3 in 6M Urea to add

O07

25

75

O15

26

74

O37

26

74

O47

38

62

O55

30

70

O77

9

91

O107

32

68

O119

16

84

O127

13

87

O142

9

91

OBLANK

100

0

There are several reagents involved in digestion. A handful of them were made by Rhonda and were viable for our use: TCEP, 1.5M Tris pH 8.8, IAA, DTT and Lys-C. Trypsin was purchased since Rhonda used all of it during her lab work.

Made 50 mM NH4HCO3 + 6M urea solution

Measured 5mL of nanopure water in a graduated cylinder, and poured into falcon tube

Weighed out 39.53 mg of ammonium bicarbonate (NH4HCO3)

Added NH4HCO3 to falcon tube, vortexed until mixed

Weighed out 3.60g Urea

Added Urea to falcon tube, vortexed until mixed

Poured falcon tube contents into graduated cylinder

Topped of contents in graduated cylinder with nanopure water up to 10 mL